Pattern formation is the activity by which embryonic cells form ordered spatial arrangements of differentiated tissues. The physical complexity of higher organisms arises during embryogenesis through the interplay of cell-intrinsic lineage and cell-extrinsic signaling. Inductive interactions are essential to embryonic patterning in vertebrate development from the earliest establishment of the body plan, to the patterning of the organ systems, to the generation of diverse cell types during tissue differentiation (Davidson, E., (1990) Development 108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al., (1992) Cell 68: 257-270). The effects of developmental cell interactions are varied. Typically, responding cells are diverted from one route of cell differentiation to another by inducing cells that differ from both the uninduced and induced states of the responding cells (inductions). Sometimes cells induce their neighbors to differentiate like themselves (homeogenetic induction); in other cases a cell inhibits its neighbors from differentiating like itself. Cell interactions in early development may be sequential, such that an initial induction between two cell types leads to a progressive amplification of diversity. Moreover, inductive interactions occur not only in embryos, but in adult cells as well, and can act to establish and maintain morphogenetic patterns as well as induce differentiation (J. B. Gurdon (1992) Cell 68:185-199).
Members of the Hedgehog family of signaling molecules mediate many important short- and long-range patterning processes during invertebrate and vertebrate development. In the fly, a single hedgehog gene regulates segmental and imaginal disc patterning. In contrast, in vertebrates, a hedgehog gene family is involved in the control of left-right asymmetry, polarity in the CNS, somites and limb, organogenesis, chondrogenesis and spermatogenesis.
The first hedgehog gene was identified by a genetic screen in the fruitfly Drosophila melanogaster (Nusslein-Volhard, C. and Wieschaus, E. (1980) Nature 287, 795-801). This screen identified a number of mutations affecting embryonic and larval development. In 1992 and 1993, the molecular nature of the Drosophila hedgehog (hh) gene was reported (C. F., Lee et al. (1992) Cell 71, 33-50), and since then, several hedgehog homologues have been isolated from various vertebrate species. While only one hedgehog gene has been found in Drosophila and other invertebrates, multiple Hedgehog genes are present in vertebrates.
The vertebrate family of hedgehog genes includes at least four members, e.g., paralogs of the single drosophila hedgehog gene. Exemplary hedgehog genes and proteins are described in PCT publications WO 95/18856 and WO 96/17924. Three of these members, herein referred to as Desert hedgehog (Dhh), Sonic hedgehog (Shh) and Indian hedgehog (Ihh), apparently exist in all vertebrates, including fish, birds, and mammals. A fourth member, herein referred to as tiggie-winkle hedgehog (Thh), appears specific to fish. Desert hedgehog (Dhh) is expressed principally in the testes, both in mouse embryonic development and in the adult rodent and human; Indian hedgehog (Ihh) is involved in bone development during embryogenesis and in bone formation in the adult; and, Shh, which as described above, is primarily involved in morphogenic and neuroinductive activities. Given the critical inductive roles of hedgehog polypeptides in the development and maintenance of vertebrate organs, the identification of hedgehog interacting proteins is of paramount significance in both clinical and research contexts.
The various Hedgehog proteins consist of a signal peptide, a highly conserved N-terminal region, and a more divergent C-terminal domain. In addition to signal sequence cleavage in the secretory pathway (Lee, J. J. et al. (1992) Cell 71:33-50; Tabata, T. et al (1992) Genes Dev. 2635-2645; Chang, D. E. et al. (1994) Development 120:3339-3353), Hedgehog precursor proteins undergo an internal autoproteolytic cleavage which depends on conserved sequences in the C-terminal portion (Lee et al. (1994) Science 266:1528-1537; Porter et al. (1995) Nature 374:363-366). This autocleavage leads to a 19 kD N-terminal peptide and a C-terminal peptide of 26-28 kD (Lee et al. (1992) supra; Tabata et al. (1992) supra; Chang et al. (1994) supra: Lee et al. (1994) supra; Bumcrot, D. A., et al. (1995) Mol. Cell. Biol. 15:2294-2303; Porter et al. (1995) supra; Ekker, S. C. et al. (1995) Curr. Biol. 5:944-955; Lai, C. J. et al. (1995) Development 121:2349-2360). The N-terminal peptide stays tightly associated with the surface of cells in which it was synthesized, while the C-terminal peptide is freely diffusible both in vitro and in vivo (Porter et al. (1995) Nature 374:363; Lee et al. (1994) supra; Bumcrot et al. (1995) supra; Mart"", E. et al. (1995) Development 121:2537-2547; Roelink, H. et al. (1995) Cell 81:445-455). Interestingly, cell surface retention of the N-terminal peptide is dependent on autocleavage, as a truncated form of HH encoded by an RNA which terminates precisely at the normal position of internal cleavage is diffusible in vitro (Porter et al. (1995) supra) and in vivo (Porter, J. A. et al. (1996) Cell 86, 21-34). Biochemical studies have shown that the autoproteolytic cleavage of the HH precursor protein proceeds through an internal thioester intermediate which subsequently is cleaved in a nucleophilic substitution. It is likely that the nucleophile is a small lipophilic molecule which becomes covalently bound to the C-terninal end of the N-peptide (Porter et al. (1996) supra), tethering it to the cell surface. The biological implications are profound. As a result of the tethering, a high local concentration of N-terminal Hedgehog peptide is generated on the surface of the Hedgehog producing cells. It is this N-terminal peptide which is both necessary and sufficient for short- and long-range Hedgehog signaling activities in Drosophila and vertebrates (Porter et al. (1995) supra; Ekker et al. (1995) supra; Lai et al. (1995) supra; Roelink, H. et al. (1995) Cell 81:445-455; Porter et al. (1996) supra; Fietz, M. J. et al. (1995) Curr. Biol. 5:643-651; Fan, C.-M. et al. (1995) Cell 81:457-465; Mart"", E., et al. (1995) Nature 375:322-325; Lopez-Martinez et al. (1995) Curr. Biol 5:791-795; Ekker, S. C. et al. (1995) Development 121:2337-2347; Forbes, A. J. et al. (1996) Development 122:1125-1135).
HH has been implicated in short- and long-range patterning processes at various sites during Drosophila development. In the establishment of segment polarity in early embryos, it has short-range effects which appear to be directly mediated, while in the patterning of the imaginal discs, it induces long range effects via the induction of secondary signals.
In vertebrates, several hedgehog genes have been cloned in the past few years. Of these genes, Shh has received most of the experimental attention, as it is expressed in different organizing centers which are the sources of signals that pattern neighboring tissues. Recent evidence indicates that Shh is involved in these interactions.
The expression of Shh starts shortly after the onset of gastrulation in the presumptive midline mesoderm, the node in the mouse (Chang et al. (1994) supra; Echelard, Y. et al. (1993) Cell 75:1417-1430), the rat (Roelink, H. et al. (1994) Cell 76:761-775) and the chick (Riddle, R. D. et al. (1993) Cell 75:1401-1416), and the shield in the zebrafish (Ekker et al. (1995) supra; Krauss, S. et al. (1993) Cell 75:1431-1444). In chick embyros, the Shh expression pattern in the node develops a left-right asymmetry, which appears to be responsible for the left-right situs of the heart (Levin, M. et al. (1995) Cell 82:803-814).
In the CNS, Shh from the notochord and the floorplate appears to induce ventral cell fates. When ectopically expressed, Shh leads to a ventralization of large regions of the mid- and hindbrain in mouse (Echelard et al. (1993) supra; Goodrich, L. V. et al. (1996) Genes Dev. 10:301-312), Xenopus (Roelink, H. et al. (1994) supra; Ruiz i Altaba, A. et al. (1995) Mol. Cell. Neurosci. 6:106-121), and zebrafish (Ekker et al. (1995) supra; Krauss et al. (1993) supra; Hammerschmidt, M., et al. (1996) Genes Dev. 10:647-658). In explants of intermediate neuroectoderm at spinal cord levels, Shh protein induces floorplate and motor neuron development with distinct concentration thresholds, floor plate at high and motor neurons at lower concentrations (Roelink et al. (1995) supra; Mart"" et al. (1995) supra; Tanabe, Y. et al. (1995) Curr. Biol. 5:651-658). Moreover, antibody blocking suggests that Shh produced by the notochord is required for notochord-mediated induction of motor neuron fates (Mart"" et al. (1995) supra). Thus, high concentration of Shh on the surface of Shh-producing midline cells appears to account for the contact-mediated induction of Floorplate observed in vitro (Placzek, M. et al. (1993) Development 117:205-218), and the midline positioning of the Floorplate immediately above the notochord in vivo. Lower concentrations of Shh released from the notochord and the Floorplate presumably induce motor neurons at more distant ventrolateral regions in a process that has been shown to be contact-independent in vitro (Yamada, T. et al. (1993) Cell 73:673-686). In explants taken at midbrain and forebrain levels, Shh also induces the appropriate ventrolateral neuronal cell types, dopaminergic (Heynes, M. et al. (1995) Neuron 15:35-44; Wang, M. Z. et al. (1995) Nature Med. 1:1184-1188) and cholinergic (Ericson, J. et al. (1995) Cell 81:747-756) precursors, respectively, indicating that Shh is a common inducer of ventral specification over the entire length of the CNS. These observations raise a question as to how the differential response to Shh is regulated at particular anteroposterior positions.
Shh from the midline also patterns the paraxial regions of the vertebrate embryo, the somites in the trunk (Fan et al. (1995) supra) and the head mesenchyme rostral of the somites (Hammerschmidt et al. (1996) supra). In chick and mouse paraxial mesoderm explants, Shh promotes the expression of sclerotome specific markers like Pax1 and Twist, at the expense of the dermamyotomal marker Pax3. Moreover, filter barrier experiments suggest that Shh mediates the induction of the sclerotome directly rather than by activation of a secondary signaling mechanism (Fan, C.-M. and Tessier-Lavigne, M. (1994) Cell 79, 1175-1186).
Shh also induces myotomal gene expression (Hammerschmidt et al. (1996) supra; Johnson, R. L. et al. (1994) Cell 79:1165-1173; Mxc3xcnsterberg, A. E. et al. (1995) Genes Dev. 9:2911-2922; Weinberg, E. S. et al. (1996) Development 122:271-280), although recent experiments indicate that members of the WNT family, vertebrate homologues of Drosophila wingless, are required in concert (Mxc3xcnsterberg et al. (1995) supra). Puzzlingly, myotomal induction in chicks requires higher Shh concentrations than the induction of sclerotomal markers (Mxc3xcnsterberg et al. (1995) supra), although the sclerotome originates from somitic cells positioned much closer to the notochord. Similar results were obtained in the zebrafish, where high concentrations of Hedgehog induce myotomal and repress sclerotomal marker gene expression (Hammerschmidt et al. (1996) supra). In contrast to amniotes, however, these observations are consistent with the architecture of the fish embryo, as here, the myotome is the predominant and more axial component of the somites. Thus, modulation of Shh signaling and the acquisition of new signaling factors may have modified the somite structure during vertebrate evolution.
In the vertebrate limb buds, a subset of posterior mesenchymal cells, the xe2x80x9cZone of polarizing activityxe2x80x9d (ZPA), regulates anteroposterior digit identity (reviewed in Honig, L. S. (1981) Nature 291:72-73). Ectopic expression of Shh or application of beads soaked in Shh peptide mimics the effect of anterior ZPA grafts, generating a mirror image duplication of digits (Chang et al. (1994) supra; Lopez-Martinez et al. (1995) supra; Riddle et al. (1993) supra) (FIG. 2g). Thus, digit identity appears to depend primarily on Shh concentration, although it is possible that other signals may relay this information over the substantial distances that appear to be required for AP patterning (100-150 xcexcm). Similar to the interaction of HH and DPP in the Drosophila imaginal discs, Shh in the vertebrate limb bud activates the expression of Bmp2 (Francis, P. H. et al. (1994) Development 120:209-218), a dpp homologue. However, unlike DPP in Drosophila, Bmp2 fails to mimic the polarizing effect of Shh upon ectopic application in the chick limb bud (Francis et al. (1994) supra). In addition to anteroposterior patterning, Shh also appears to be involved in the regulation of the proximodistal outgrowth of the limbs by inducing the synthesis of the fibroblast growth factor FGF4 in the posterior apical ectodermal ridge (Laufer, E. et al. (1994) Cell 79:993-1003; Niswander, L. et al. (1994) Nature 371:609-612).
The close relationship between Hedgehog proteins and BMPs is likely to have been conserved at many, but probably not all sites of vertebrate Hedgehog expression. For example, in the chick hindgut, Shh has been shown to induce the expression of Bmp4, another vertebrate dpp homologue (Roberts, D. J. et al. (1995) Development 121:3163-3174). Furthermore, Shh and Bmp2, 4, or 6 show a striking correlation in their expression in epithelial and mesenchymal cells of the stomach, the urogenital system, the lung, the tooth buds and the hair follicles (Bitgood, M. J. and McMahon, A. P. (1995) Dev. Biol. 172:126-138). Further, Ihh, one of the two other mouse Hedgehog genes, is expressed adjacent to Bmp expressing cells in the gut and developing cartilage (Bitgood and McMahon (1995) supra).
Recent evidence suggests a model in which Ihh plays a crucial role in the regulation of chondrogenic development (Roberts et al. (1995) supra). During cartilage formation, chondrocytes proceed from a proliferating state via an intermediate, prehypertrophic state to differentiated hypertrophic chondrocytes. Ihh is expressed in the prehypertrophic chondrocytes and initiates a signaling cascade that leads to the blockage of chondrocyte differentiation. Its direct target is the perichondrium around the Ihh expression domain, which responds by the expression of Gli and Patched (Ptc), conserved transcriptional targets of Hedgehog signals (see below). Most likely, this leads to secondary signaling resulting in the synthesis of parathyroid hormone-related protein (PTHrP) in the periarticular perichondrium. PTHrP itself signals back to the prehypertrophic chondrocytes, blocking their further differentiation. At the same time, PTHrP represses expression of Ihh, thereby forming a negative feedback loop that modulates the rate of chondrocyte differentiation.
Patched was originally identified in Drosophila as a segment polarity gene, one of a group of developmental genes that affect cell differentiation within the individual segments that occur in a homologous series along the anterior-posterior axis of the embryo. See Hooper, J. E. et al. (1989) Cell 59:751; and Nakano, Y. et al. (1989) Nature 341:508. Patterns of expression of the vertebrate homologue of patched suggest its involvement in the development of neural tube, skeleton, limbs, craniofacial structure, and skin.
Genetic and functional studies demonstrate that patched is part of the hedgehog signaling cascade, an evolutionarily conserved pathway that regulates expression of a number of downstream genes. See Perrimon, N. (1995) Cell 80:517; and Perrimon, N. (1996) Cell 86:513. Patched participates in the constitutive transcriptional repression of the target genes; its effect is opposed by a secreted glycoprotein, encoded by hedgehog, or a vertebrate homologue, which induces transcriptional activation. Genes under control of this pathway include members of the Wnt and TGF-beta families.
Patched proteins possess two large extracellular domains, twelve transmembrane segments, and several cytoplasmic segments. See Hooper, supra; Nakano, supra; Johnson, R. L. et al. (1996) Science 272:1668; and Hahn, H. et al. (1996) Cell 85:841. The biochemical role of patched in the hedgehog signaling pathway is unclear. Direct interaction with the hedgehog protein has, however, been reported (Chen, Y. et al. (1996) Cell 87:553), and patched may participate in a hedgehog receptor complex along with another transmembrane protein encoded by the smoothened gene. See Perrimon, supra; and Chen, supra.
The human homologue of patched was recently cloned and mapped to chromosome 9q22.3. See Johnson, supra; and Hahn, supra. This region has been implicated in basal cell nevus syndrome (BCNS), which is characterized by developmental abnormalities including rib and craniofacial alterations, abnormalities of the hands and feet, and spina bifida.
Sporadic tumors also demonstrated a loss of both functional alleles of patched. Of twelve tumors in which patched mutations were identified with a single strand conformational polymorphism screening assay, nine had chromosomal deletion of the second allele and the other three had inactivating mutations in both alleles (Gailani, supra). The alterations did not occur in the corresponding germline DNA.
Most of the identified mutations resulted in premature stop codons or frame shifts. Lench, N. J., et al., Hum. Genet. 1997 October; 100(5-6): 497-502. Several, however, were point mutations leading to amino acid substitutions in either extracellular or cytoplasmic domains. These sites of mutation may indicate functional importance for interaction with extracellular proteins or with cytoplasmic members of the downstream signaling pathway.
The involvement of patched in the inhibition of gene expression and the occurrence of frequent allelic deletions of patched in BCC support a tumor suppressor function for this gene. Its role in the regulation of gene families known to be involved in cell signaling and intercellular communication provides a possible mechanism of tumor suppression.
The present invention makes available methods and reagents for inhibiting aberrant growth states resulting from activation of the hedgehog signaling pathway, such as hedgehog gain-of-function, by contacting the cell with an agent, such as a small molecule, in a sufficient amount to reverse or control the aberrant growth state, e.g., to agonize a normal ptc pathway, antagonize a normal hedgehog pathway, or antagonize smoothened activity.
In one aspect, the invention pertains to a method for inhibiting an altered growth state of a cell having a hedgehog gain-of-function phenotype by contacting the cell with a hedgehog antagonist in a sufficient amount to inhibit the altered growth state, wherein the hedgehog antagonist is a organic molecule represented in the general formula (I): 
wherein, as valence and stability permit,
R1 and R2, independently for each occurrence, represent H, lower alkyl, xe2x80x94(CH2)naryl (substituted or unsubstituted), or xe2x80x94(CH2)nheteroaryl (substituted or unsubstituted);
L, independently for each occurrence, is absent or represents xe2x80x94(CH2)n-alkyl, -alkenyl-, -alkynyl-, xe2x80x94(CH2)nalkenyl-, xe2x80x94(CH2)nalkynyl-, xe2x80x94(CH2)nO(CH2)pxe2x80x94, xe2x80x94(CH2)nNR2(CH2)pxe2x80x94, xe2x80x94(CH2)nS(CH2)pxe2x80x94, xe2x80x94(CH2)nalkenyl(CH2)pxe2x80x94, xe2x80x94(CH2)nalkynyl(CH2)pxe2x80x94, xe2x80x94O(CH2)nxe2x80x94, xe2x80x94NR2(CH2)nxe2x80x94, or xe2x80x94S(CH2)nxe2x80x94;
X1 and X2 are selected, independently, from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94(R8)Nxe2x80x94N(R8)xe2x80x94, xe2x80x94ON(R8)xe2x80x94, a heterocycle, or a direct bond between L and Y1 or Y2, respectively;
Y1 and Y2 are selected, independently, from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94C(xe2x95x90NCN)xe2x80x94, xe2x80x94P(xe2x95x90O)(OR2)xe2x80x94, a heteroaromatic group, or a direct bond between X1 and Z1 or X2 and Z2, respectively;
Z1 and Z2 are selected, independently, from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94R8Nxe2x80x94NR8xe2x80x94, xe2x80x94ONR8xe2x80x94, a heterocycle, or a direct bond between Y1 or Y2, respectively, and L;
R8, independently for each occurrence, represents H, lower alkyl, xe2x80x94(CH2)naryl (substituted or unsubstituted), xe2x80x94(CH2)nheteroaryl (substituted or unsubstituted), or two R8 taken together form a 4- to 8-membered ring, together with the atoms to which they are attached, which ring may include one or more carbonyls;
p represents, independently for each occurrence, an integer from 0 to 10; and
n, individually for each occurrence, represents an integer from 0 to 10.
In certain embodiments, R1 represents a substituted or unsubstituted aryl or heteroaryl group. In certain embodiments, at least one of X1xe2x80x94Y1xe2x80x94Z1 and X2xe2x80x94Y2xe2x80x94Z2 taken together represents a urea or an amide. In certain embodiments, R1 represents either a fused cycloalkyl-aryl or cycloalkyl-heteroaryl system.
In certain embodiments, the hedgehog antagonist inhibits hedgehog-mediated signal transduction with an ED50 of 1 mM or less, 1 xcexcM or less, 100 nM or less, 10 nM or less, or 1 nM or less. The cell may be contacted with the hedgehog antagonist in vitro or in vivo. In certain embodiments, the hedgehog antagonist is administered as part of a therapeutic or cosmetic application, such as regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, or regulation of skin and hair growth.
In another aspect, the invention relates to a method for inhibiting an altered growth state of a cell having a hedgehog gain-of-function phenotype by contacting the cell with a hedgehog antagonist in a sufficient amount to inhibit the altered growth state, wherein the hedgehog antagonist is a organic molecule represented in the general formula (II): 
wherein, as valence and stability permit,
R1 and R2, independently for each occurrence, represent H, lower alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or unsubstituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or unsubstituted);
L, independently for each occurrence, is absent or represents xe2x80x94(CH2)n-alkyl, -alkenyl-, -alkynyl-, xe2x80x94(CH2)nalkenyl-, xe2x80x94(CH2)nalkynyl-, xe2x80x94(CH2)nO(CH2)pxe2x80x94, xe2x80x94(CH2)nNR2(CH2)pxe2x80x94, xe2x80x94(CH2)nS(CH2)pxe2x80x94, xe2x80x94(CH2)nalkenyl(CH2)pxe2x80x94, xe2x80x94(CH2)nalkynyl(CH2)pxe2x80x94, xe2x80x94O(CH2)nxe2x80x94, xe2x80x94NR2(CH2)nxe2x80x94, or xe2x80x94S(CH2)nxe2x80x94;
X is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94(R8)Nxe2x80x94N(R8)xe2x80x94, xe2x80x94ON(R8)xe2x80x94, a heterocycle, or a direct bond between L and Y;
Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94C(xe2x95x90NCN)xe2x80x94, xe2x80x94P(xe2x95x90O)(OR2)xe2x80x94, a heteroaromatic group, or a direct bond between X and Z;
Z is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94R8Nxe2x80x94NR8xe2x80x94, xe2x80x94ONR8xe2x80x94, a heterocycle, or a direct bond between Y and L;
R8, independently for each occurrence, represents H, lower alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or unsubstituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or unsubstituted), or two R8 taken together form a 4- to 8-membered ring, together with the atoms to which they are attached, which ring may include one or more carbonyls;
W represents a substituted or unsubsituted aryl or heteroaryl ring fused to the pyrimidone ring;
p represents, independently for each occurrence, an integer from 0 to 10; and
n, individually for each occurrence, represents an integer from 0 to 10.
In certain embodiments, R1 represents a substituted or unsubstituted aryl or heteroaryl group. In certain embodiments, Xxe2x80x94Yxe2x80x94Z taken together represents a urea or an amide. In certain embodiments, W is a substituted or unsubstituted benzene ring. In certain embodiments, X represents a diazacarbocycle. In certain embodiments, R2 represents a substituted or unsubstituted aryl or heteroaryl group. In certain embodiments, R8, independently for each occurrence, is selected from H and lower alkyl. In certain embodiments, X is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, and a direct bond; Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, and xe2x80x94S(O2)xe2x80x94; and Z is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, and a direct bond, such that at least one of X and Z is present. In certain embodiments, at least one of X and Z is present. In certain embodiments, Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, and xe2x80x94S(O2)xe2x80x94.
In certain embodiments, the hedgehog antagonist inhibits hedgehog-mediated signal transduction with an ED50 of 1 mM or less, 1 xcexcM or less, 100 nM or less, 10 nM or less, or 1 nM or less. The cell may be contacted with the hedgehog antagonist in vitro or in vivo. In certain embodiments, the hedgehog antagonist is administered as part of a therapeutic or cosmetic application, such as regulation of neural tissues, bone and cartilage formation and repair, regulation of spermatogenesis, regulation of smooth muscle, regulation of lung, liver and other organs arising from the primative gut, regulation of hematopoietic function, or regulation of skin and hair growth.
In yet another embodiment, the invention provides a pharmaceutical preparation comprising a sterile pharmaceutical excipient and a compound represented by the general formula (I): 
wherein, as valence and stability permit,
R1 and R2, independently for each occurrence, represent H, lower alkyl, xe2x80x94(CH2)naryl (substituted or unsubstituted), or xe2x80x94(CH2)nheteroaryl (substituted or unsubstituted);
L, independently for each occurrence, is absent or represents xe2x80x94(CH2)n-alkyl, -alkenyl-, -alkynyl-, xe2x80x94(CH2)nalkenyl-, xe2x80x94(CH2)nalkynyl-, xe2x80x94(CH2)nO(CH2)pxe2x80x94, xe2x80x94(CH2)nNR2(CH2)pxe2x80x94, xe2x80x94(CH2)nS(CH2)pxe2x80x94, xe2x80x94(CH2)nalkenyl(CH2)pxe2x80x94, xe2x80x94(CH2)nalkynyl(CH2)pxe2x80x94, xe2x80x94O(CH2)nxe2x80x94, xe2x80x94NR2(CH2)nxe2x80x94 or xe2x80x94S(CH2)nxe2x80x94;
X1 and X2 are selected, independently, from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94(R8)Nxe2x80x94N(R8)xe2x80x94, xe2x80x94ON(R8)xe2x80x94, a heterocycle, or a direct bond between L and Y1 or Y2, respectively;
Y1, and Y2 are selected, independently, from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94C(xe2x95x90NCN)xe2x80x94, xe2x80x94P(xe2x95x90O)(OR2)xe2x80x94, a heteroaromatic group, or a direct bond between X1 and Z1 or X2 and Z2, respectively;
Z1 and Z2 are selected, independently, from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94R8Nxe2x80x94NR8xe2x80x94, xe2x80x94ONR8xe2x80x94, a heterocycle, or a direct bond between Y1 or Y2, respectively, and L;
R8, independently for each occurrence, represents H, lower alkyl, xe2x80x94(CH2)naryl (substituted or unsubstituted), xe2x80x94(CH2)nheteroaryl (substituted or unsubstituted), or two R8 taken together form a 4- to 8-membered ring, together with the atoms to which they are attached, which ring may include one or more carbonyls;
p represents, independently for each occurrence, an integer from 0 to 10; and
n, individually for each occurrence, represents an integer from 0 to 10.
In certain embodiments, R1 represents a substituted or unsubstituted aryl or heteroaryl group. In certain embodiments, at least one of X1xe2x80x94Y1xe2x80x94Z1 and X2xe2x80x94Y2xe2x80x94Z2 taken together represents a urea or an amide. In certain embodiments, R1 represents either a fused cycloalkyl-aryl or cycloalkyl-heteroaryl system.
In another aspect, the invention relates to a pharmaceutical preparation comprising a sterile pharmaceutical excipient and a compound represented by the general formula (II): 
wherein, as valence and stability permit,
R1 and R2, independently for each occurrence, represent H, lower alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or unsubstituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or unsubstituted);
L, independently for each occurrence, is absent or represents xe2x80x94(CH2)n-alkyl, -alkenyl-, -alkynyl-, xe2x80x94(CH2)nalkenyl-, xe2x80x94(CH2)nalkynyl-, xe2x80x94(CH2)nO(CH2)pxe2x80x94, xe2x80x94(CH2)nNR2(CH2)pxe2x80x94, xe2x80x94(CH2)nS(CH2)pxe2x80x94, xe2x80x94(CH2)nalkenyl(CH2)pxe2x80x94, xe2x80x94(CH2)nalkynyl(CH2)pxe2x80x94, xe2x80x94O(CH2)nxe2x80x94, xe2x80x94NR2(CH2)nxe2x80x94, or xe2x80x94S(CH2)nxe2x80x94;
X is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94(R8)Nxe2x80x94N(R8)xe2x80x94, xe2x80x94ON(R8)xe2x80x94, a heterocycle, or a direct bond between L and Y;
Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94C(xe2x95x90NCN)xe2x80x94, xe2x80x94P(xe2x95x90O)(OR2)xe2x80x94, a heteroaromatic group, or a direct bond between X and Z;
Z is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94R8Nxe2x80x94NR8xe2x80x94, xe2x80x94ONR8xe2x80x94, a heterocycle, or a direct bond between Y and L;
R8, independently for each occurrence, represents H, lower alkyl, aryl (substituted or unsubstituted), aralkyl (substituted or unsubstituted), heteroaryl (substituted or unsubstituted), or heteroaralkyl (substituted or unsubstituted), or two R8 taken together form a 4- to 8-membered ring, together with the atoms to which they are attached, which ring may include one or more carbonyls;
W represents a substituted or unsubsituted aryl or heteroaryl ring fused to the pyrimidone ring;
p represents, independently for each occurrence, an integer from 0 to 10; and
n, individually for each occurrence, represents an integer from 0 to 10.
In certain embodiments, R1 represents a substituted or unsubstituted aryl or heteroaryl group. In certain embodiments, Xxe2x80x94Yxe2x80x94Z taken together represents a urea or an amide. In certain embodiments, W is a substituted or unsubstituted benzene ring. In certain embodiments, X represents a diazacarbocycle. In certain embodiments, R2 represents a substituted or unsubstituted aryl or heteroaryl group. In certain embodiments, R8, independently for each occurrence, is selected from H and lower alkyl. In certain embodiments, X is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, and a direct bond; Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, and xe2x80x94S(O2)xe2x80x94; and Z is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, and a direct bond, such that at least one of X and Z is present. In certain embodiments, at least one of X and Z is present. In certain embodiments, Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, and xe2x80x94S(O2)xe2x80x94.
In still another aspect, the invention relates to a compound having the general structure of Formula II: 
wherein, as valence and stability permit,
R1 and R2, independently for each occurrence, represent H, lower alkyl, xe2x80x94(CH2)naryl (e.g., substituted or unsubstituted), or xe2x80x94(CH2)nheteroaryl (e.g., substituted or unsubstituted);
L, independently for each occurrence, is absent or represents xe2x80x94(CH2)n-alkyl, -alkenyl-, -alkynyl-, xe2x80x94(CH2)nalkenyl-, xe2x80x94(CH2)nalkynyl-, xe2x80x94(CH2)nO(CH2)pxe2x80x94, xe2x80x94(CH2)nNR2(CH2)pxe2x80x94, xe2x80x94(CH2)nS(CH2)pxe2x80x94, xe2x80x94(CH2)nalkenyl(CH2)pxe2x80x94, xe2x80x94(CH2)nalkynyl(CH2)pxe2x80x94, xe2x80x94O(CH2)nxe2x80x94, xe2x80x94NR2(CH2)nxe2x80x94, or xe2x80x94S(CH2)nxe2x80x94;
X is xe2x80x94NHxe2x80x94;
Y is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94C(xe2x95x90S)xe2x80x94, xe2x80x94S(O2)xe2x80x94, xe2x80x94S(O)xe2x80x94, xe2x80x94C(xe2x95x90NCN)xe2x80x94, xe2x80x94P(xe2x95x90O)(OR2)xe2x80x94, a heteroaromatic group, or a direct bond between X and Z;
Z is selected from xe2x80x94N(R8)xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Sexe2x80x94, xe2x80x94Nxe2x95x90Nxe2x80x94, xe2x80x94ONxe2x95x90CHxe2x80x94, xe2x80x94R8Nxe2x80x94NR8xe2x80x94, xe2x80x94ONR8xe2x80x94, a heterocycle, or a direct bond between Y and L;
R8, independently for each occurrence, represents H, lower alkyl, xe2x80x94(CH2)naryl (e.g., substituted or unsubstituted), xe2x80x94(CH2)nheteroaryl (e.g., substituted or unsubstituted), or two R8 taken together may form a 4- to 8-membered ring, e.g., with X1 and Z2 or X2 and Z2, which ring may include one or more carbonyls;
W represents a substituted or unsubsituted aryl or heteroaryl ring fused to the pyrimidone ring;
p represents, independently for each occurrence, an integer from 0 to 10, preferably from 0 to 3; and
n, individually for each occurence, represents an integer from 0 to 10, preferably from 0 to 5.
In certain embodiments, L adjacent to X represents -(unbranched lower alkyl)-. In certain embodiments, R1 represents an unsubstituted aryl or heteroaryl ring, or an aryl or heteroaryl ring substituted with substituents selected from H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, hydroxyl, (unbranched alkyl-Oxe2x80x94), silyloxy, amino, nitro, thiol, amino, imino, amido, phosphoryl, phosphonate, phosphine, carbonyl, carboxyl, carboxamide, anhydride, silyl, thioether, alkylsulfonyl, arylsulfonyl, sulfoxide, selenoether, ketone, aldehyde, ester, or xe2x80x94(CH2)mxe2x80x94R8.
In certain embodiments, R1 represents an unsubstituted aryl or heteroaryl ring, or an aryl or heteroaryl ring substituted with substituents selected from H, halogen, cyano, alkyl, alkenyl, alkynyl, aryl, nitro, thiol, imino, amido, carbonyl, carboxyl, anhydride, thioether, alkylsulfonyl, arylsulfonyl, ketone, aldehyde, and ester. In certain embodiments, R1 represents an unsubstituted aryl or heteroaryl ring, or an aryl or heteroaryl ring substituted with substituents selected from H, halogen, cyano, alkyl, alkenyl, alkynyl, nitro, amido, carboxyl, anhydride, alkylsulfonyl, ketone, aldehyde, and ester. In certain embodiments, R2 is a substituted or unsubstituted aryl or heteroaryl ring. In certain embodiments, Xxe2x80x94Yxe2x80x94Zxe2x80x94 represents an amide or urea linkage. In certain embodiments, R8 represents H for all occurrences. In certain embodiments, R2 is a substituted or unsubstituted aryl or heteroaryl ring. In certain embodiments, L is absent adjacent to R1.
In certain embodiments, the compound has a structure selected from the structures depicted in FIGS. 32j, k, and l, 
In yet another aspect, the invention relates to a method for preparing a bicyclic compound, comprising heating a reaction mixture comprising a compound having a structure of Formula X with a carboxylic acid anhydride according to the scheme: 
wherein
W represents an aryl or heteroaryl ring, and
R10 represents substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, or heteroaralkyl, and
wherein the anhydride has a structure of (R10CH2Cxe2x95x90O)2O wherein Rxe2x80x2 is CH2R10.
In certain embodiments, the reaction mixture consists essentially of the compound of Formula X and the carboxylic acid anhydride at the start of the reaction.
In still another aspect, the invention provides a method for preparing an amine, comprising contacting a compound having a structure of Formula XIII with an amine having a structure of H2NR12 according to the scheme: 
wherein W represents an aryl or heteroaryl ring;
R1 represents H, lower alkyl, xe2x80x94(CH2)naryl, or xe2x80x94(CH2)nheteroaryl;
L is absent or represents xe2x80x94(CH2)n-alkyl, -alkenyl-, -alkynyl-, xe2x80x94(CH2)nalkenyl-, xe2x80x94(CH2)nalkynyl-, xe2x80x94(CH2)nO(CH2)pxe2x80x94, xe2x80x94(CH2)nNR2(CH2)pxe2x80x94, xe2x80x94(CH2)nS(CH2)pxe2x80x94, (CH2)nalkenyl(CH2)pxe2x80x94, xe2x80x94(CH2)nalkynyl(CH2)pxe2x80x94, xe2x80x94O(CH2)nxe2x80x94, xe2x80x94NR2(CH2)nxe2x80x94, or xe2x80x94S(CH2)nxe2x80x94;
Yxe2x80x2 represents a halogen;
R10 represents substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, or heteroaralkyl; and
R12 represents a lower alkyl group or a silyl group,
wherein the amine and the compound having a structure of Formula XIII are combined with a polar solvent comprising less than about 50% water.
In certain embodiments, the polar solvent comprises an alcohol. In certain embodiments, the alcohol is selected from methanol, ethanol, propanol, isopropanol, butanol, isobutanol, t-butanol, sec-butanol, ethylene glycol, and 1,3-propanediol.
In another aspect, the invention provides a method for preparing a compound, comprising performing steps according to the scheme: 
wherein
step (A) comprises reacting a compound having a structure of Formula X, wherein W represents a substituted or unsubstituted aryl or heteroaryl ring, such as a benzene ring, having an amino group and a carboxylic acid group in adjacent (ortho) positions, with an acylating agent having the formula R10CH2C(xe2x95x90O)Xxe2x80x2, wherein R10, independently for each occurrence, represents substituted or unsubstituted alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, aralkyl, or heteroaralkyl, and Xxe2x80x2 represents a halogen or xe2x80x94OC(xe2x95x90O)CH2R10, under conditions that produce a compound having a structure of Formula XI;
step (B) comprises reacting a compound having a structure of Formula XI with an amine having the formula R1LNH2, wherein R1 and L are as defined above, under conditions that result in a compound having a structure of Formula XII;
step (C) comprises reacting a compound having a structure of Formula XII with a halogenating agent, such as chlorine, bromine, iodine, N-bromosuccinimide, N-chlorosuccinimide, N-iodosuccinimide, ClBr, IBr, ClI, or a reagent that generates a halogen radical (such as Cl., Br., or I.) under conditions that result in a compound having a structure of Formula XIII, wherein Yxe2x80x2 represents a halogen such as Cl, Br, or I;
step (D) comprises reacting a compound having a structure of Formula XIII with an amine having the formula H2NR12, wherein R12 represents a lower alkyl group or a silyl group, such as a trialkylsilyl, triarylsilyl, dialkylarylsilyl, or diarylalkylsilyl group, under conditions that result in a compound having a structure of Formula XIV; and
step (E) comprises reacting a compound having a structure of Formula XIV with a terminating group having a structure of R2Vxe2x80x2 to produce a compound having a structure of Formula XX, wherein R2 is as defined above, and Vxe2x80x2 represents a functional group selected from ZC(xe2x95x90W)Cl, ZC(xe2x95x90W)Br, isocyanate, isothiocyanate, ZC(xe2x95x90W)WC(xe2x95x90W)ZR2, ZSO2Cl, ZSO2Br, ZSOCl, ZSOBr, or an activated acylating moiety prepared in situ.
In still another aspect, the invention provides a method for preparing a compound, comprising performing steps according to the scheme: 
wherein
step (A) comprises reacting a compound having a structure of Formula XV, wherein Pxe2x80x2 represents H or a protecting group, W represents a substituted or unsubstituted aryl or heteroaryl ring, such as a benzene ring, having an amino group and a carboxylic acid or ester group in adjacent (ortho) positions, with an acylating agent having the formula PXLC(xe2x95x90O)Xxe2x80x2, wherein X and L are as defined above, P represents a protecting group, and Xxe2x80x2 represents a halogen, xe2x80x94OC(xe2x95x90O)LXP, or a functional group generated by reacting a carboxyl group with an activating agent, such as a carbodiimide (e.g., diisopropylcarbodiimide, dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, etc.), phosphorous-based reagents (such as BOP-Cl, PyBROP, etc.), oxalyl chloride, phosgene, triphosgene, carbonyldiimidazole, or any other reagent that reacts with a carboxylic acid group resulting in a reactive intermediate having an increased susceptibility, relative to the carboxylic acid, towards coupling with an amine, under conditions that produce a compound having a structure of Formula XVI;
step (B) comprises deprotecting the ester of a compound having a structure of Formula XVI to produce a carboxylic acid having a structure of Formula XVII, if necessary;
step (C) comprises reacting a compound having a structure of Formula XVII with an amine having the formula R1LNH2, wherein R1 and L are as defined above, under conditions that result in a compound having a structure of Formula XVIII;
step (D) comprises removing the protecting group P from a compound having a structure of Formula XVIII to generate a compound having a structure of Formula XIX;
step (E) comprises reacting a compound having a structure of Formula XIX with a terminating group having a structure of R2Yxe2x80x2 to produce a compound having a structure of Formula XXI, wherein R2 is as defined above, and Yxe2x80x2 represents a functional group selected from ZC(xe2x95x90W)Cl, ZC(=W)Br, isocyanate, isothiocyanate, ZC(xe2x95x90W)WC(xe2x95x90W)ZR2, ZSO2Cl, ZSO2Br, ZSOCl, ZSOBr, or an active acylating moiety prepared in situ.